Project Summary The goal of this proposal is to define the mechanisms controlling dynamic assembly and function of cellular actin structures in a living, developing animal. Recent advances in single molecule in vitro TIRF microscopy have made major contributions to our understanding of the molecular mechanisms controlling actin and microtubule rearrangements. However, the field is lagging behind in the integration of these in vitro mechanisms into a physiological context by testing them in animal models. To fill this gap, we will use a combined `top-down' and `bottom-up' strategy, bringing together the genetic and live imaging power of Drosophila with quantitative in vitro reconstitution and single molecule analysis. Specifically, we focus on a group of interacting actin assembly factors, which includes Adenomatous polyposis coli proteins 1 and 2 (APC1 and APC2) and the formin Diaphanous (Dia), and how they work together to build a striking array of long actin cables in Drosophila nurse cells at a specific stage of oogenesis. Our central hypothesis is that APC1, APC2, and Dia each make a mechanistically distinct and essential contribution to actin cable formation, and that the proper spatial and temporal pattern of cable assembly arises from these proteins working in concert under tight regulation by interactions with microtubules and microtubule-associated proteins (e.g., EB1 and CLIP-170), and regulated by GSK3 kinase. By directly visualizing these mechanisms at the single molecule level in vitro, and using this information to guide rigorous multi-faceted tests in living Drosophila ovaries, we will arrive at an unprecedented level of mechanistic understanding of actin assembly and microtubule-actin cross-talk in the living animal. The specific aims are: (1) Determine how actin cable arrays are assembled during oogenesis by tightly controlled multi-component mechanisms involving collaborative actin filament nucleation and elongation factors; and (2) Define the mechanisms by which microtubules trigger actin cable emergence and interact with growing cables to guide their morphogenesis and their contact with the nucleus.